Scrub typhus is a life-threatening disease caused Orientia tsutsugamushi, a LPS-negative bacterium that replicates preferentially in endothelial cells and monocytes. Approximately one million people are infected every year; about one third of the world's population is at risk of infection. There is no effective vaccine for this infection; information on disease pathogenesis and T cell immunity is limited. To address these challenges in this research field, we have developed C57BL6 mouse models that can mimic pathological features of human scrub typhus. We found that lethal infection was linked to type 1-biased immune responses, which correlated with excessive cellular apoptosis and vascular damage in multiple organs. In addition, we have identified the top vaccine candidates (p47 and p56) and their protective fragments from extensive studies of a panel of Orientia major antigens/formulas against homologues and heterologous challenges in outbred mice. The goal of this study is to examine how T cell responses control protective and pathogenic immune responses during infection with O. tsutsugamushi Karp, the most prevalent strain for human infections. Our central hypothesis is that appropriate T cell priming, through multi-variant vaccines or non-lethal infection, will help elicit a balanced immunity against bacterial spread, acute tissue damage and severe scrub typhus. Aim 1 will test that a multi-variant DNA vaccine platform can protect B6 mice against severe scrub typhus through the stimulation of a balanced activation of T- and B-cell immunity. At different stages of immunization and lethal challenge, we will evaluate tissue bacterial loads, pathological changes, as well as the kinetics and levels of host immune responses. Aim 2 will test the hypothesis that a progressive loss of T cells, especially Ag-specific CD8+ T cells, is the underlying mechanism for endothelial damage and contributing factor for lethal infection. We will use synthetic peptides that represent four predicted T epitopes and recombinant proteins to estimate cytokine production in re-call assays, as well as the frequency and fate of cytokine-secreting T cells. In vitro data will be integrated with those from immunostaining of apoptotic cells in the spleen and other organs. This vaccine- based, T cell-focused study endorses synergy among research teams (each with unique expertise); it utilizes the state-of-art ABSL3/BSL3 facilities at UTMB. The long-term goal of this study is to examine the potential of a multi-variant DNA vaccine platform and protective cellular immunity for the control of scrub typhus. Experiential evidence for a balanced T-cell activation in an immune-protected host would be particularly important, given the current status and gap in this neglected emerging disease. This timely study will have a broad implication for other intracellular pathogens.